I have a problem in gene expression.
It is about 9 months that I am unsuccessful in expression.
my target gene is Eukaryotic and 405bp.
And I optimized all of conditions that are necessary for expression Eukaryotic gene in bacterial cell like codons usage, GC content , mRNA folding and etc.
Gene expression vectors that I used for cloning are
PET 28, 32, 22 and PGEX 4T-1 vector.
the cloning Confirmed by PCR and digest .
OD: different condition(0.4-1) 620nm
IPTG: different condition (0.3-1)Mmol/lit
incubation Temperatures: different condition(22-37) C
Incubation times: 2-24h
competent cell for transformation BL21?DE3.
It should be noted that when the empty vector(PET32) was transformed in to competent cell with above condition was expressed.
However, the cloned gene is not expressed.